Mechanisms and Function of Autophagy in Cancer Summary The overall goal of this research proposal is to elucidate the molecular mechanisms of autophagy and its role in cancer. Autophagy is a lysosome-mediated, stress-responsive catabolic pathway that plays important role in both normal biology and disease. Although the core molecular pathway of autophagy has been relatively well delineated, how this pathway is precisely regulated to accomplish specific and sometime seemingly counterintuitive function is not clear. For example, what is the mechanism that enables ?autophagy-addicted? cancer cells to possess both intact mTOR activity and high levels of basal autophagy, considering mTOR is a bona fide inhibitor of autophagy? What is the role of autophagy in determining various cell fates such as cell death, senescence, and stemness, which are all relevant to tumorigenesis? What therapeutic implication can we learn from the answers to these basic biological questions concerning autophagy? In the previous funding cycle, we have observed potent anticancer effect of autophagy-inhibition in cellular and mouse models for multiple cancer types, including glioblastoma (GBM) and pancreatic ductal adenocarcinoma (PDAC). We discovered that a specific protein phosphatase 2A (PP2A) complex can be stimulated to enhance autophagy initiation through dephosphorylating the autophagy-initiating protein kinase complex, the ULK1 complex, thus counteracting the autophagy-suppressing activity of mTOR. Importantly, our preliminary results indicate that PP2A is also involved in the activation of TFEB transcriptional factor, a master regulator of autophagy gene expression and lysosomal biogenesis. Therefore, via regulating the ULK1 complex and TFEB, PP2A modulates both initiation and potency of autophagy. Intriguingly, we also found that autophagy and TFEB regulate GBM cell senescence under clinically relevant conditions. Based on these preliminary studies, in this grant, we will further investigate the molecular basis of autophagy and its role in cancer, by focusing on the following two aims: (1) the mechanisms by which protein phosphatases regulate TFEB, and the functional impact of such regulation on autophagy and autophagy-addicted cancer cells; and (2) the role of autophagy and TFEB in cancer cell senescence. To achieve these aims, we will conduct experiments that employ a combination of approaches, including standard cellular, molecular and biochemical approaches, animal modeling, and more specialized technologies such as quantitative mass spectrometry and deep sequencing. Success of the proposed study will lead to an in-depth mechanistic understanding of the regulation and function of autophagy in normal biology and cancer biology, and might provide insights into precise targeting of autophagy for cancer treatment. The proposal will also shed light on the functional interplay of autophagy with other important biological processes, including lysosomal biogenesis and senescence.